Monoclonal antibody CRA against a fraction of actin from cress roots recognizes its antigen in different plant species

Abstract

The globular cytoskeletal protein G-actin was isolated from the crude extract of soluble proteins from cress (Lepidium sativum L.) roots. The crude extract was loaded onto a deoxyribonuclease (DNase) I-affinity chromatography column and subsequently eluted with EGTA and urea. The fraction eluted with 2 mM EGTA was characterized by molecular weight determination, binding to DNase I, isoelectric focusing, and immunoblotting. These samples clearly showed one main 43,000 dalton protein with a pI value between 5.5 and 5.7. This polypeptide is an isoform of actin. It was stained using commercially available monoclonal and polyclonal actin antibodies. We used the EGTA fraction as plant actin antigen to produce a monoclonal cress root actin antibody. Antibodies (CRA) showed specific labelings on Western immunoblots against a 43,000 dalton protein of the cress root crude extract. Under the fluorescence microscope CRA detected actin in fixed statenchyma cells of cress roots. This antibody also demonstrated intact bundles of actin filaments in unfixed internodal cells of Chara australis. On the basis of these results we concluded that we had obtained a new monoclonal antibody (CRA) against actin from cress roots. We also found a cress root actin-binding protein antibody (CRAB) showing a filament staining pattern in internodal cells of Chara.